Aberrant DNA methylation is a dominant mechanism in MDS progression to AML

Abstract

Myelodysplastic syndromes (MDSs) are clonal hematologic disorders that frequently represent an intermediate disease stage before progression to acute myeloid leukemia (AML). As such, study of MDS/AML can provide insight into the mechanisms of neoplastic evolution. In 184 patients with MDS and AML, DNA methylation microarray and high-density single nucleotide polymorphism array (SNP-A) karyotyping were used to assess the relative contributions of aberrant DNA methylation and chromosomal deletions to tumor-suppressor gene (TSG) silencing during disease progression. Aberrant methylation was seen in every sample, on average affecting 91 of 1505 CpG loci in early MDS and 179 of 1505 loci after blast transformation (refractory anemia with excess blasts [RAEB]/AML). In contrast, chromosome aberrations were seen in 79% of early MDS samples and 90% of RAEB/AML samples, and were not as widely distributed over the genome. Analysis of the most frequently aberrantly methylated genes identified FZD9 as a candidate TSG on chromosome 7. In patients with chromosome deletion at the FZD9 locus, aberrant methylation of the remaining allele was associated with the poorest clinical outcome. These results indicate that aberrant methylation can cooperate with chromosome deletions to silence TSG. However, the ubiquity, extent, and correlation with disease progression suggest that aberrant DNA methylation is the dominant mechanism for TSG silencing and clonal variation in MDS evolution to AML.

Figure 1

Validation of the methylation array. (A) Methylation profiles of replicate samples show high reproducibility. Data shown are methylation β-values of 1505 CpG sites in which 1 and 0 represent high and low methylation levels, respectively. Each dot represents a CpG site. (B) Methylation array data correlate with methylation-enriched PCR results. Methylation-enriched PCR was used to measure the methylation status of CpG sites of DBC1 and DCC genes in bone marrow mononuclear cells from 3 healthy control donors and 5 patients with AML. ■ indicates the methylation-enriched PCR results represented as fold change compared with the results in the patient with AML marked with an asterisk. indicates the array average methylation values (β-value) for the same CpG sites.

Figure 2

Hypermethylated genes in leukemic cell lines belong to many functional groups. (A) Myeloid leukemia cell lines have increased CpG site methylation levels (β-values), and a high frequency of aberrantly methylated CpG sites, compared with normal CD34⁺ hematopoietic cells. The vertical boxes delineate the interquartile range in β-values; T bars, 95% range of values. The horizontal line in the boxes indicates the median values and the plus sign indicates the mean. The asterisk indicates significant β-value difference with CD34⁺ cells in a Tukey Studentized Range t test. The numbers below the graph are the number of aberrantly methylated loci (compared with normal CD34⁺ control). The text explains why the frequency of aberrant methylation may be underestimated if only β-values are analyzed. (B) Heat-map analysis showing the methylation β-values of 58 CpG loci that were concordantly hypermethylated in all 6 leukemia cell lines. Red, black, and green correspond to high, medium, and low methylation levels, respectively. Genes were grouped according to their functions: group I, apoptosis and cell cycle; group II, cell development; group III, cell proliferation and adhesion; group IV, cell differentiation; group V, chromosome architecture; group VI, signal transduction; and group VII, other.

Figure 3

Aberrant promoter methylation correlates with disease evolution. (A) Patients with RAEB/AML show a higher average and median methylation (β-value; P = .001) and a higher number of aberrantly methylated sites (P < .001) than those with low-risk MDS (aberrant methylation was defined as a methylation β-value for a CpG site that was significantly greater (P < .001) than the methylation β-value for the corresponding site in the group of normal bone marrow controls). The number of aberrantly methylated CpG sites in patients with complex cytogenetic abnormalities (defined as 3 or more abnormalities by standard metaphase karyotyping) is significantly higher (P = .05) than in patients without complex cytogenetic abnormalities; however, the average methylation level (β-value) was not significantly different (P = .23). Each dot represents the average array methylation level in a patient; the horizontal dashed line indicates the mean value for the patient group. Numbers below the graph represent the average number of aberrantly methylated CpG sites (of 1505 sites analyzed) in each patient group. Normal indicates normal whole bone marrow; CD34⁺ are normal CD34⁺ selected hematopoietic cells. (B) The number of concordantly hypermethylated sites by patient group, defined as CpG loci that were aberrantly methylated in more than 50% of patients with RAEB/AML, low-risk MDS, and CMML, respectively. Each circle represents a patient group, and the overlapping areas represent common aberrantly methylated genes. (C) CpG sites that became hypermethylated during evolution of RA to RCMD. Each dot represents a CpG site. The hypermethylated CpG sites are listed. (D,E) Concordantly hypermethylated loci in patients with low-risk MDS and RAEB/AML, respectively. Each dot represents a CpG site. Genes shown more than once represent those with more than one hypermethylated loci. The percentage refers to the proportion of patients with aberrant methylation at the specified locus.

Figure 4

Aberrant DNA hypermethylation was detected much more frequently than chromosome aberrations. (A) Proportion of patients with chromosome aberrations detected by standard metaphase karyotyping or SNP-A by patient group. (B) Idiogram representing the chromosomal localization of concordantly hypermethylated loci (loci hypermethylated in more than 50% of patients with low-risk MDS and RAEB/AML). Chromosomes are numbered at the top. Gene names are indicated beside each hypermethylated locus. Genes marked with a box are hypermethylated in both low-risk MDS and AML/RAEB. Genes shown more than once represent those with more than one hypermethylated locus. (C) On a chromosome by chromosome basis, the proportion of patients with aberrant methylation detected on that chromosome was higher than the proportion of patients with a chromosome aberration involving that chromosome.

Figure 5

Aberrant methylation at the FZD9 locus is a predictor of prognosis in MDS/AML. (A) FZD9 hypermethylation is associated with decreased 12-month survival. (B) FZD9 methylation levels in individual patients classified by disease group. Each dot represents the methylation β-value of the FZD9 CpG site in an individual patient. The average methylation β-value of this CpG site for each patient group is indicated by the horizontal line. (C) FZD9 methylation is inversely correlated to FZD9 transcript expression. represents the degree of FZD9 methylation in the sample; ■, the FZD9 expression levels determined by semiquantitative RT-PCR, defined as fold change compared with the control marked with an asterisk. (D) FZD9 CpG methylation is not part of a wider hypermethylation at its chromosome 7 locus. The height of the vertical bars represents the frequency of aberrant hypermethylation of the CpG sites designated along the horizontal line. The space between bars marked with 0 indicates analyzed CpG sites that were not hypermethylated in any patient. (E) Regions of chromosome deletion and UPD, detected by SNP-A, that involves the FZD9 locus. FZD9 is designated by the vertical red bar. Red lines depict single SNP signal intensity, whereas green lines present an average value of SNP signal intensity. The horizontal purple bar represents areas of chromosome deletion, whereas the horizontal blue bar represents areas of loss of heterozygosity through UPD. (F) Chromosomal deletion or duplication of an FZD9 allele, combined with aberrant methylation of the remaining allele, is associated with a worse prognosis than either abnormality alone.